Increasing Concentration of Inhaled Saline with or without Amiloride Effect on Mucociliary Clearance in Normal Subjects

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1 Increasing Concentration of Inhaled Saline with or without Amiloride Effect on Mucociliary Clearance in Normal Subjects Namita Sood, William D. Bennett, Kirby Zeman, James Brown, Carla Foy, Richard C. Boucher, and Michael R. Knowles Division of Pulmonary and Critical Care Medicine, Cystic Fibrosis/Pulmonary Research Center, and Center for Environmental Medicine and Lung Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and Division of Pulmonary and Critical Care Medicine, Ohio State University, Columbus, Ohio of ASL, concentration of mucin macromolecules, and slowing of MCC (7, 8). In vivo, aerosolized hypertonic saline or man- nitol increases the rate of mucus clearance in patients with cystic fibrosis, perhaps reflecting partial restoration of ASL volume (11, 12). In contrast to cystic fibrosis, patients with systemic pseudohypoaldosteronism (PHA) have loss-of-func- tion mutations in the epithelial Na channel (ENaC) and an absence of airway epithelial Na absorption, which is associated with an increased volume of isotonic ASL (13, 14). Adults with PHA appear to compensate for the excess ASL by accelerated rates of clearance of liquid (MCC) from the airway surface (14). No previous study has systematically tested the relation- ship between an acute increase in ASL volume and the rate of MCC in normal subjects. The goal of the current study was to study the effect of maneuvers predicted to acutely increase the volume of ASL on MCC rates in normal subjects. Our strategy was to measure MCC rates with isotopic mark- ers delivered after airway surfaces were dosed by inhalation with increasing amounts of NaCl in aerosolized 0.12, 0.9, and 7% saline. We further tested the hypothesis that pretreatment with inhaled amiloride, an Na channel blocker, might increase the magnitude of inhaled NaCl induced changes in MCC by preventing Na (and ASL volume) absorption over the MCC measurement interval (4, 15). Mucociliary clearance is determined by ciliary activity and rheology of airway surface liquid. To test the hypothesis that mucociliary clearance would increase after inhalation of an osmotically active agent that would increase the volume of airway surface liquid, we measured mucociliary clearance in 16 normal subjects after inhalation of varying tonicities of saline alone, and after pretreatment with a Na channel blocker (amiloride). Subjects inhaled vehicle (0.12% saline) or amiloride, followed by inhalation of 0.12, 0.9, or 7% saline. Subsequently, mucociliary clearance rates were mea- sured by scintigraphy of inhaled 99m Tc Fe 2 O 3. Mucociliary clearance of whole and peripheral lung was increased (approximately twofold) after inhalation of increasing concentrations of saline (p 0.04). Pretreatment with amiloride increased mucociliary clearance rates (approximately twofold) after inhalation of 0.12 and 0.9% saline (p 0.05), but not 7% saline. The rates of mucociliary clear- ance by pretreatment with amiloride and 7% saline alone (approxi- mately 1.4% per minute) approached the rapid mucociliary clearance rates (approximately 2.0% per minute) reported in systemic pseudohypoaldosteronism, which has loss-of-function mutations of the epithelial Na channel and an increased volume of airway surface liquid. We conclude that maneuvers that increase the volume of airway surface liquid are associated with increased rates of mucociliary clearance in normal subjects. Keywords: mucociliary clearance; airway surface liquid; epithelial Na channel; aerosolized NaCl Mucociliary clearance (MCC) rates depend on ciliary activity coupled to the volume and rheological properties of airway surface liquid (ASL) (1), and rates of MCC can be upregulated for airway host defense. Under normal circumstances, there is cephalad movement of the ASL secondary to ciliary activity (2). The volume (depth) of ASL is regulated by isotonic volume transport, with active Na absorption as the dominant basal ion transport activity of the airway surface epithelia (2 8). In vitro, the acute addition of NaCl expands ASL height (9), improves mucus rheologic properties (elas- ticity and viscosity), and accelerates mucus transport rates (9, 10). In cystic fibrosis there is accelerated absorption of NaCl from airway surfaces, which leads to reduced volume (depth) (Received in original form April 7, 2002; accepted in final form October 21, 2002) This study was supported by the following grants: CFF RDP R026, NIH SCOR 5-P50- HL , and GCRC RR Correspondence and requests for reprints should be addressed to Namita Sood, M.D., Division of Pulmonary and Critical Care Medicine, Ohio State University, 201, Heart & Lung Research Institute, 473 West 12th Avenue, Columbus, OH sood-1@medctr.osu.edu Am J Respir Crit Care Med Vol 167. pp , 2003 Originally Published in Press as DOI: /rccm OC on October 31, 2002 Internet address: METHODS Subjects Sixteen normal subjects completed this protocol. The first cohort (Group 1; Table 1) had seven subjects (six females) with a mean age of 25 years. The second cohort (Group 2) had 10 subjects (5 females) with a mean age of 29.9 years; 1 subject (female, aged 22 years) was excluded based on predetermined criteria because of variation in ratio of central to peripheral (C/P) radionuclide counts. The study was approved by the University of North Carolina IRB and informed consent was obtained. Techniques Drug preparation, storage, and administration. Dry powder amiloride (Merck, West Point, PA) was dissolved in 0.12% saline as a stock (10 2 M). Radioisotopic aerosol labeling, generation, and administration. The aerosol (4 m mass median aerodynamic diameter [MMAD]) was formed from 2% (by weight) colloidal suspension of 99m Tc-Fe 2 O 3 by a spinning disk aerosol generator to produce dry iron oxide particles (16). Protocol Subjects were studied over 4 days in a randomized, double-blind, crossover design. To limit isotope exposure, two groups were studied and exposed to a common treatment (0.9% saline). Before isotope was administered, subjects inhaled solutions of vehicle or amiloride, then different concentrations of saline, for up to 15 minutes (or until

2 Sood, Bennett, Zeman, et al.: Inhaled NaCl and Mucus Clearance 159 TABLE 1. AEROSOLIZED DOSING REGIMENS saline is similar to basal (no treatment) clearance in healthy Group 1* (n 7) Group 2 (n 9) subjects, and does not represent a stimulated rate of MCC (17, 18). In general, the whole lung (Figure 2) and peripheral lung 0.12% saline/0.12% saline ND (data not shown) MCC curves revealed an early period of rapid Amiloride/0.12% saline ND clearance, which then plateaued to a slower rate. This change 0.12% saline/0.9% saline 0.12% saline/0.9% saline in rate could reflect either a true slowing of clearance, and/or Amiloride/0.9% saline Amiloride/0.9% saline ND 0.12% saline/7% saline reduced availability of clearable isotopic marker in conducting ND Amiloride/7% saline airways, over time. To quantitate the initial rate of rapid isotope clearance, we Definition of abbreviation: ND not done. calculated the breakpoint in the rate of clearance, which reflects Aerosolized dosing regimens were administered in randomized and blinded the time-shift from a phase of rapid to apparent slower clearfashion on four separate days to two groups of study subjects. *C/P ance (see Methods). The breakpoint in the rate of whole and C/P peripheral lung MCC for all study days was determined to be 20 minutes. This analysis indicated that the predominant effect of the different aerosolized solutions on rates of MCC occurred within the first 20 minutes after deposition of isotope-labeled sputter ; see Figure 1), using a PARI LC Star nebulizer with an iron oxide particles. MMAD of approximately 4 m (Figure 1). The lowest concentration The whole lung clearance rates (%/minute) as a function of saline (0.12%; hypotonic) was selected because the rate of MCC is of concentration of inhaled saline with or without amiloride not stimulated in normal subjects after inhalation of 0.12% saline, i.e., pretreatment over the first 20 minutes for subjects from both the rate of MCC after hypotonic saline (alone) is the basal rate of groups are summarized in Figure 2C. For inhaled saline without MCC in healthy subjects (17, 18). Subsequently, subjects inhaled an aerosol (4 m MMAD; geometric SD 1.25) of insoluble 99m Tc-Fe amiloride pretreatment, increasing masses of NaCl, delivered 2 O 3 (19, 20). An initial deposition scan was recorded and scanning was by aerosols with varying concentrations of NaCl, increased the performed for 120 minutes (2-minute images every 10 minutes) to rate of MCC when tested for all study subjects (p 0.04, analysis measure particle clearance. Subjects returned at 24 hours after deposition of variance). There was little effect for 0.9% saline to increase for a 30 minute scan. MCC as compared with 0.12% saline (p 0.19, nonpaired t test), Data Analysis whereas 7% saline clearly increased MCC as compared with 0.12 saline (p 0.03). Pretreatment with amiloride increased The approach for measurement and analysis is well-used in our labs the rates of MCC observed after inhalation of 0.12% (p 0.02) (17, 21). In brief, particle clearance after inhalation is initially rapid, but or 0.9% saline (p ), but not 7% saline. Further, with slows after the first minutes (Figure 2). This pattern of clearance amiloride pretreatment, the rates of MCC after inhalation of (initially rapid, with later slowing) is also seen in measures of baseline 0.12 and 0.9% saline (i.e., %/minute) were similar to that (no treatment) MCC (22). Initial comparisons between different aerosol treatments were made by repeated-measures analysis of variance of observed for 7% saline alone. clearance over the initial 20 minutes for each day. Comparisons between The peripheral lung clearance rates (%/minute) over the first doses of saline (0.12% versus 0.9% saline) were assessed by nonpaired 20 minutes for all subjects are shown in Figure 2D. For inhaled t tests, whereas comparisons of pretreatment with amiloride were as- saline aerosols without amiloride pretreatment, there was a tensessed by two-tailed paired t tests. Significance was set at p 0.05 for dency for increasing masses of inhaled NaCl, i.e., increased two-tailed analysis. Unless otherwise specified, data are presented as concentrations of saline, to increase the rate of MCC (p 0.12, means SD. analysis of variance). Comparisons of MCC after inhalation of Modeling (Estimating) the Relative Volume (Depth) of ASL different concentrations of saline revealed no effect of 0.9% saline to increase MCC as compared with 0.12% saline, but 7% after Aerosolized Saline saline increased peripheral MCC compared with 0.12% saline. These estimates used a model that has been validated by comparison Again, pretreatment with amiloride increased peripheral MCC with experimental (total and regional deposition) lung deposition mearates after inhalation of 0.12 and 0.9% saline (p 0.05), but surements in humans over a large range of particle sizes (23 26). not 7% saline. After pretreatment with amiloride, the rates of RESULTS MCC (approximately 1%/minute) were similar for all concentrations of inhaled saline. Whole lung clearances of 99m Tc iron oxide particles are shown We also analyzed the MCC data over longer-term intervals for different aerosolized solutions in the first group (n 7; (0 to 60 and 120 minutes). For whole lung clearance, pretreat- Figure 2A) and the second group (n 9; Figure 2B) of subjects. ment with inhaled amiloride systematically increased the fraction The pattern and rate of clearance after inhalation of 0.12/0.12% of isotope cleared after inhalation of 0.12% saline (p Figure 1. Design of protocol for each study day. After background lung scan, subjects inhaled vehicle (0.12% saline) or amiloride (10 mm in a nebulizer), followed by different doses of NaCl. Isotope (Tc99mlabeled Fe 2 O 3 particles) was then inhaled as a radio-marker for measurement of mucociliary clearance over 2 hours, with a follow-up scan 24 hrs later.

3 160 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 2. Mucociliary clearance (retention versus time) for different aerosolized solutions. A shows whole lung clearance for the first group of subjects (n 7; C/P ), and B shows whole lung clearance for the second group of subjects (n 9; C/P ); amiloride pretreatment increased the rates of whole lung mucociliary clearance for 0.12 and 0.9% saline over 2 hours (p 0.001), but did not enhance the effects of 7% saline (see text for details). C shows whole lung mucociliary clearance rates over the initial 20 minutes for different treatment regimens for all subjects. Increasing doses of saline (0.12 to 0.9 to 7%) increased the rates of MCC (p 0.04; analysis of variance). Pretreatment with amiloride increased rates of whole lung MCC for 0.12 and 0.9% saline (*p 0.02 compared with 0.12% alone; p , compared with 0.9% alone), but not 7% saline. D shows peripheral lung mucociliary clearance rates over the initial 20 minutes for different treatment regimens for all subjects. Increasing doses of saline (0.12 to 0.9 to 7%) tended to increase the rates of peripheral MCC (p 0.l2; analysis of variance). Pretreatment with amiloride increased rates of peripheral lung MCC for 0.12 and 0.9% saline (*p 0.02 compared with 0.12% alone; p 0.05 compared with 0.9% alone), but not 7% saline. for 60 and 120 minutes) and 0.9% saline (p for 120 The aerosol inhalations were well tolerated by all subjects, minutes), when compared with pretreatment with inhalation of and there were no episodes of bronchospasm or other untoward vehicle (Figure 2A). In contrast, amiloride pretreatment did effects. To assess the effect of study design on the results, we not increase the net clearance induced by 7% saline when determined the role of several covariates to any observed differ- compared with pretreatment with vehicle (Figure 2B). Similar ences in clearance for the different treatment study days. Overall, patterns were seen for net clearance over 60 and 120 minutes only subject and treatment were significant (p 0.05) predictors from the peripheral lung regions (data not shown). of the variation in 20-minute clearance rate. For 0.9 and 7% At 24 hours (Figures 2A and 2B), pretreatment with amiloride saline, treatment interaction with C/P ratio was significant (p tended to increase net clearance when compared with 0.12 and 0.03) for predicting 20-minute clearance rate, which implies that 0.9% saline, alone (p 0.09 for whole lung; p for the difference in 20-minute clearance rate between treatments peripheral lung), but amiloride had no effect on net clearance was greatest as C/P increased. There was no evidence for a role as compared with 7% saline aerosols with vehicle pretreatment for cough frequency over the period of interest, treatment order, or for either whole or peripheral lung. interactions between these two variables and treatment (p 0.4).

4 Sood, Bennett, Zeman, et al.: Inhaled NaCl and Mucus Clearance 161 ing estimates may be better correlated with conditions after pretreatment with amiloride, which blocks Na (and volume) absorption. Figure 3. Changes in depth of airway surface liquid (ASL) after inhalation of saline, as estimated by modeling techniques (see METHODS). A shows the increase in depth of ASL (left ordinate) after inhalation of 0.9% saline versus the fraction of aerosol particles deposited (right ordinate) on the surfaces of proximal to distal generations of conducting airways (abscissa). B shows the predicted increase in depth of ASL after inhalation of 0.12 and 0.9 and 7% saline. This study demonstrated that maneuvers designed to acutely increase the volume (depth) of ASL are associated with increased rates of MCC in normal subjects (Figure 2). In general, increasing the amount (mass) of salt deposited on airway surfaces by increasing the concentration of NaCl in aerosols increased MCC. Similarly, pretreatment with amiloride to promote retention of inhaled NaCl on airway surfaces increased the effect of saline aerosols to raise MCC rates. We designed our protocols to focus on the effects of added salt on ciliary-dependent, rather than cough-dependent, Fe 2 O 3 (mucus) clearance. To remove the potential contribution of cough clearance in response to the different saline amiloride aerosol solutions, subjects were exposed to the different aerosol solutions before radiolabeled isotopic marker was given. With this protocol, there were no significant episodes of coughing after the inhalation of the tracer. Only four subjects experienced any cough and a multivariate analysis failed to show any effect of cough on MCC. There also appeared to be no change in airway caliber after delivery of the test aerosol that would bias estimation of MCC based on tracer deposition, as a comparable deposition of the inhaled radiolabel was achieved over the four study days as reflected by relatively uniform C/P ratios. Thus, there were no artifacts of cough-clearance or regional differences in tracer deposition inherent in this protocol design, which may have limited quantitative interpretation of the effects of saline aerosols on MCC in other studies, where cough occurred during nebulization of (hypertonic) saline or amiloride (11, 12). In this study, there was little effect of inhaled isotonic (0.9%) saline to stimulate MCC as compared with a hypotonic saline (0.12). This result may reflect the fact that the difference in inhaled salt mass between a 0.12% saline aerosol (1.2 mg) and a 0.9% saline aerosol (9 mg) is relatively small (approximately 8 mg). In contrast, the mass of delivered salt is relatively large with 7% saline (70 mg), perhaps accounting for the large increase in MCC after inhalation of 7% as compared with 0.12 and 0.9% saline. Indeed, this hypothesis is consistent with the model analyses comparing salt deposition with increased ASL height (Fig- ure 3B). Interestingly, amiloride administered before inhalation of smaller doses of saline (0.12 or 0.9%) induced twofold increases in rates of MCC over the comparable saline doses with vehicle pretreatment for whole and peripheral lung (see Figures 2C and 2D). The effect of amiloride pretreatment to increase the MCC response to 0.12 and 0.9% saline (Figure 2) could reflect the capacity of amiloride to conserve all salt added to airway surfaces before and during the interval of the MCC measurement. However, because the quantity of added salt with 0.12% saline is relatively small, and there is little predicted effect on ASL height (Figure 3), the increase in MCC rates effected by amiloride may reflect the drug s effect to block basal ASL volume absorption (4, 27). The failure of amiloride to potentiate the effect of 7% saline inhalation on MCC could reflect the fact that the block of basal absorption is small relative to the very large mass of salt, and consequently, ASL volume, which is added to the airway surface by the concentrated (7%) salt solution. It is also likely that the addition of such a large mass of salt led to an acute increase in the ASL, resulting in a clearance of most of the radioisotope by 20 minutes. From the MCC protocol used in this study, it is difficult to clearly define the duration of the effect on MCC after acute To approximate the relative change in depth of ASL along the tracheobronchial tree, we estimated the increase in the depth of ASL after acute inhalation of saline from modeling (see Methods). In Figure 3A, we plotted estimates of particle deposition fraction after inhalation of 0.9% saline (right ordinate) based on the parameters of the aerosol droplet size and respiratory flow rates. We then estimated the volume of ASL added from the volume of 0.9% saline aerosol deposited and the estimated surface area of each airway generation (left ordinate). Although the deposition fraction of particles is relatively similar along different generations of the conducting airways, the model sug- gests that the greatest relative increase in the depth of ASL occurs in proximal (third generation) bronchi, whereas the small- est change is in more distal (12th 16th generation) bronchioles. These differences in estimated depth of ASL added largely reflect the relative surface areas of proximal (small surface) versus distal (large surface) conducting airways. In Figure 3B, we plotted the relative estimated change in added ASL depth as a function of the concentration of inhaled saline. As can be seen, the change in ASL depth is not a function of the fold-change in saline concentration (i.e., eightfold for 0.9 versus 0.12%, and 7 versus 0.9%, respectively), but rather a direct function of the mass of NaCl added to the airway surface by the aerosols of different concentrations of NaCl (see Figure 3B). We recognize that saline is deposited over 15 minutes in our research protocol in humans, and absorption of Na (and volume) is taking place over that interval, in the absence of amiloride. Thus, these model- DISCUSSION

5 162 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL addition of salt amiloride to airway surfaces (Figure 2). The the normal subjects in the study led to ASL volume addition effect of inhaled saline to rapidly accelerate MCC appeared to with selective hydration/swelling of the mucus layer and an accel- last only 20 minutes, which (if true) could reflect rapid absorption eration of MCC rates, although the precise mechanism to link of Na (and volume) and a short duration of effect on MCC. This these events is not known. The greater change in rates of MCC interpretation is compatible with studies of well-differentiated in whole lung versus peripheral regions after similar aerosol human bronchial epithelial cell cultures that observed rapid treatments (Figures 2C and 2D), which parallel the predicted ( 12 min) absorption of an equivalent quantity of saline (9%) regional changes in ASL volume (Figure 3B), are also congruent (9, 15). However, the strategy to pretreat with amiloride and with this hypothesis. to block volume absorption would be predicted to extend the In summary, maneuvers that are predicted to acutely increase duration of effect at least 30 to 60 minutes beyond the 20 minutes the volume of ASL are associated with acceleration of MCC rates observed here, as demonstrated previously in vitro (9). Interest- in normal subjects. These findings are consistent with previous ingly, in vivo measurements in patients with PHA also demon- studies that demonstrated the short-term effect of inhaled hyperstrated that the duration of rapid radiotracer clearance, i.e., tonic saline and/or amiloride to increase mucociliary clearance MCC, lasted only 20 minutes (14). Because patients with PHA in patients with cystic fibrosis (4, 11, 12, 28, 29). The development have a genetic loss-of-function defect of Na channels with a of long-acting osmotic agents, and/or drugs that block volume constitutive defect in volume absorption, the duration (20 min- absorption (or stimulate liquid secretion) in the airways, has utes) of rapid rate of particle clearance (MCC) in these patients therapeutic potential for the treatment of patients with reduced cannot reflect the variables in this study (i.e., retention time of volume of ASL, including cystic fibrosis. salt or amiloride on airway surfaces), but likely reflects a techni- Acknowledgment : The authors express their appreciation to Lisa Brown for graphcal limitation of the MCC protocol. The simplest hypothesis is ical and editorial assistance. that there is reduced availability of clearable isotopic marker over time, related in part to inhaled Fe 2 O 3 particles entering a References second compartment on airway surfaces (e.g., macrophages) that 1. Wanner A, Salathe M, O Riordan TG. 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